In vitro functional assays of KREL1 full-length and domain truncated mutants with and without KREPA2. (A) Structural representation of the KREL1 constructs used including full-length (FL), NTD, NTD_VLR and the CTD provided in trans. (B) Three-step ligation assay of KREL1 FL vs. NTD, with and without KREPA2 WT. T4Rnl2 was used as a positive control, and KREL1 K87A was used as a negative control. Identities of RNA species are depicted to the left of the gels. The RNA being visualized contains a CY5 label. (C) Ligase auto-adenylylation assay, representative of step 1 of ligation, of KREL1 FL and domain, truncated mutants with and without KREPA2. A “+” symbol represents reactions with domains provided in trans. The KREL1–AMP product is radiolabeled with α-32P represented by an asterisk. (D) Preadenylated ligation assay, representative of step 3 of ligation, of KREL1 FL vs. NTD with and without KREPA2. T4Rnl2 was used as a positive control. The RNA being visualized contains a CY5 label.
